Pilot-scale process development and scale up for antifungal production

A pilot-scale fermentation was developed for an antifungal compound produced by a filamentous fungus. Replacement of galactose with lactose (20-fold cost savings) and a threefold phosphate reduction (15 to 5 g/L) improved productivity 2.5-fold. Addition of supplements—glycine, cobalt chloride, and trace elements—resulted in a further twofold productivity increase, greater process robustness, and less foaming which reduced antifoam addition tenfold (30 to <3 mL/L). Mid-cycle lactose limitations were addressed by raising initial lactose levels (40 to 120 g/L) resulting in another twofold productivity increase. Overall, peak titers increased tenfold from 45 ± 9 to 448 ± 39 mg/L, and productivities improved from 3 to 25 mg/L day. Despite its high productivity, process scale up was challenged by high broth viscosity (5,000–6,000 cP at 16.8 s⁻¹). Gassed power requirements at the 600 L scale (4.7 kW/1,000 L) exceeded available power at the 15,000 L scale (3.0 kW/1,000 L), and broth transfer to the downstream isolation facility was hindered. Mid-cycle broth dilution with up to five 10 vol% additions of 12 wt% lactose solution or whole medium-reduced viscosity three- to fivefold (1,000–1,500 cP at 16.8 s⁻¹), gassed power within scale-up limits (2.5 kW/1,000 L), and peak titer by up to 45%. The process was scaled up to the 15,000 L working volume based on constant aeration rate (vvm) and peak impeller tip speed, raising superficial velocities at similar shear. This strategy maximized mass transfer rates at target gassed power per unit volume levels, and along with controlled broth viscosity, precluded multiple dilution additions. A final titer of 333 mg/L with one dilution addition was achieved, somewhat lower than expected, likely owing to inhibition from some unmeasured volatile compound (not believed to be carbon dioxide) during an extended period of high back-pressure in the early production phase.     

Acute antihypertensive action of nitroxides in the spontaneously hypertensive rat

Tempol is an amphipathic radical nitroxide (N) that acutely reduces blood pressure (BP) and heart rate (HR) in the spontaneously hypertensive rat (SHR). We investigated the hypothesis that the response to nitroxides is determined by SOD mimetic activity or lipophilicity. Groups (n = 6-10) of anesthetized SHRs received graded intravenous doses of Ns: tempol (T), 4-amino-tempo (AT), 4-oxo-tempo (OT), 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT-1), 3-carbamoyl-proxyl (3-CP), or 3-carboxy-proxyl (3-CTPY). Others received native or liposomal (L) Cu/Zn SOD. T and OT are uncharged, AT is positively charged and cell-permeable, and CAT-1 is positively charged and cell-impermeable. 3-CP and 3-CTPY have five-member pyrrolidine rings, whereas T, AT, OT, and CAT-1 have six-member piperidine rings. T and AT reduced mean arterial pressure (MAP) similarly (-48 +/- 2 mmHg and -55 +/- 8 mmHg) but more (P < 0.05) than OT and CAT-1. 3-CP and 3-CTPY were ineffective. The group mean change in MAP with piperidine Ns correlated with SOD activity (r = -0.94), whereas their ED(50) correlated with lipophilicity (r = 0.89). SOD and L-SOD did not lower BP acutely but reduced it after 90 min (-32 +/- 5 and -31 +/- 6 mmHg; P < 0.05 vs. vehicle). Pyrrolidine nitroxides are ineffective antihypertensive agents. The antihypertensive response to piperidine Ns is predicted by SOD mimetic action, and the sensitivity of response is by hydrophilicity. SOD exerts a delayed hypotensive action that is not enhanced by liposome encapsulation, suggesting it must diffuse to an extravascular site.     

Respiratory symptoms and disease characteristics as predictors of pulmonary function abnormalities in patients with rheumatoid arthritis: an observational cohort study

Introduction  

Lung involvement is a common extra-articular manifestation of rheumatoid arthritis (RA) that confers significant morbidity and mortality. The objective of the present study is to assess which respiratory symptoms and patient and disease characteristics are most highly associated with pulmonary function test (PFT) abnormalities in an RA patient cohort without clinical cardiovascular disease.     

Breadth of Human Immunodeficiency Virus-Specific Neutralizing Activity in Sera: Clustering Analysis and Association with Clinical Variables

Induction of antibodies that neutralize a broad range of human immunodeficiency virus type 1 (HIV-1) isolates is a major goal of vaccine development. To study natural examples of broad neutralization, we analyzed sera from 103 HIV-1-infected subjects. Among progressor patients, 20% of sera neutralized more than 75% of a panel of 20 diverse viral isolates. Little activity was observed in sera from long-term nonprogressors (elite controllers). Breadth of neutralization was correlated with viral load, but not with CD4 count, history of past antiretroviral use, age, gender, race/ethnicity, or route of exposure. Clustering analysis of sera by a novel method identified a statistically robust subgrouping of sera that demonstrated broad and potent neutralization activity.Eliciting neutralizing antibodies (NAbs) against human immunodeficiency virus type 1 (HIV-1) is likely to be crucial for an optimally effective vaccine. To date, the antibodies elicited by vaccines have had weak activity against a limited spectrum of HIV-1 strains (10, 22, 33). However, many HIV-infected patients make NAbs, and a small fraction make extremely potent NAbs with broad cross-reactivity (3, 4, 9, 26, 29, 32). Understanding how a broadly reactive NAb response develops in some HIV-1-infected patients, and what viral epitopes are targeted, may provide important clues for vaccine design (18). The prevalence and clinical parameters associated with broadly reactive NAbs in serum have been the subject of much recent interest (11, 28, 29). We therefore examined the potency and breadth of neutralization in a large cohort of patients, compared breadth with clinical and demographic variables, and used clustering analysis to discern patterns in serum reactivity to diverse isolates.In a previous study (9), we screened HIV-infected patient sera for neutralizing activity against a panel of five viral isolates, using a TZM-bl Env pseudovirus neutralization assay. We also established a more robust 20-viral-isolate panel that included 10 clade B, 5 clade A, and 5 clade C Env pseudoviruses (9, 16-18). In order to evaluate the prevalence of neutralization breadth in a more quantitative manner, we studied 103 patient sera against all 20 viruses. All patients participated in National Institutes of Health clinical protocols, were infected for at least 1 year, and were antiretroviral (ARV) naïve or had been off ARVs for at least 3 months at the time of sampling. All patients were presumed to be infected with clade B virus based on locations of current and former residences. Eighty-one of the patients were included in the previously published analysis (9). Twenty-five patients were long-term nonprogressors (LTNP; also called elite controllers) from the cohort described in references 23 and 24, who typically maintain a viral load (VL) of <50 RNA copies/ml and a stable CD4⁺ T-cell count without ARV therapy; this group had a median CD4⁺ T-cell count of 850 cells/μl and a median time since HIV diagnosis of 13.5 years. The other 78 patients had a median viral load of 4,931 RNA copies/ml, a median CD4⁺ T-cell count of 534 cells/μl, and a median of 12.5 years since diagnosis. This patient group includes both typical progressors and patients without CD4⁺ T-cell decline (referred to in prior reports as slow progressors). In our previous analysis (9), we found no differences in neutralization breadth between typical and slow progressors; therefore, for the purposes of this report, both patient groups are analyzed together and collectively referred to as progressors. Dates of diagnosis but not of seroconversion were available. We calculated both the 50% and 80% inhibitory doses (ID₅₀ and ID₈₀, respectively) for each isolate using the TZM-bl assay as described in reference 31.Among progressor patients with readily detectible viremia, wide ranges of serum neutralization potency and breadth were observed (Fig. ​(Fig.1A).1A). Using a cutoff ID₅₀ of ≥100, we found that these sera neutralized a median of 10.5 (interquartile range [IQR], 5 to 14) out of 20 isolates. A total of 20% of these sera were broadly reactive, neutralizing at least 15 of 20 isolates on our panel. However, 50% of the sera neutralized 10 or fewer isolates, with several sera having very low activity despite years of untreated viremia. In contrast, sera from LTNP, with <50 copies of HIV RNA/ml plasma, had little neutralization activity, with a median of only 1 of 20 isolates neutralized with an ID₅₀ of ≥100 (IQR, 0 to 2.5) (Fig. ​(Fig.1B).1B). The range of neutralization activity in this group was similar to the lowest end of values for progressor patients. This observation concurs with previous data from our laboratory and others which show that, compared to patients with higher levels of viremia, LTNP make weak NAb responses, perhaps due to reduced antigenic stimulation of B cells (2, 9, 15, 20, 27). To ensure that we were measuring serum-mediated viral neutralization, we also incorporated ID₈₀ values into the analysis. Using a cutoff of both an ID₅₀ of ≥100 and ID₈₀ of ≥15, sera of progressor patients neutralized a median of 9 (IQR, 2.8 to 11) isolates, with 15% of sera neutralizing >75% of viruses. In contrast, among LTNP the median was 0 (IQR, 0 to 1).Open in a separate windowFIG. 1.Neutralization of 20 isolates by patient sera. (A and B) ID₅₀ value against each of 20 isolates in the TZM-bl assay is plotted for each patient. The red line indicates an ID₅₀ of 100. Input dilution was 1:10; if no neutralization was observed, the ID₅₀ is plotted as 5. (A) Progressors. (B) LTNP. (C) Viral load (RNA copies/ml plasma) versus geometric mean titer for each patient''s serum. Red circles, LTNP; black circles, progressor patients.Clinical and demographic data for all patients were compared to neutralization breadth. Two parameters were used to quantify breadth for each serum: the geometric mean ID₅₀ against the 20 isolates and the number neutralized with both an ID₅₀ of ≥100 and an ID₈₀ of ≥15. The associations between breadth and clinical covariates were tested using nonparametric methods (Spearman''s rho, Wilcoxon rank-sum, or Kruskal-Wallis test). A Bonferroni correction was used as follows to adjust for the multiple comparisons: reported P values are multiplied by 10 from the original P values and are considered significant if they are below 0.05. Viral load had a modest positive association with breadth, as measured both by geometric mean ID₅₀ (P < 0.001; r = 0.68) (Fig. ​(Fig.1C)1C) and by the number of isolates neutralized (P < 0.001; r = 0.63). When the analysis was restricted to progressor patients, this relationship still held (P = 0.008 and r = 0.37 for geometric mean ID₅₀; P = 0.050 and r = 0.31 for number neutralized). CD4⁺ T-cell count showed a modest negative correlation with breadth by both measures (P = 0.025 and r = −0.29 for geometric mean ID₅₀; P = 0.031 and r = −0.29 for number neutralized), but this relationship was not significant when LTNP were excluded from the analysis. Years since diagnosis, HLA class II alleles, risk group, history of using ARVs, race, ethnicity, gender, and age were not associated with breadth by either measure. Thus, the only strong predictor of breadth found in this cohort is viral load.To find patterns of neutralization reactivity in this data set, and determine potential common specificities of neutralization, we performed a clustering analysis based on the ID₅₀ values for the 103 sera and 20 isolates. This analysis is shown as a heat map in Fig. ​Fig.2,2, in which darker red colors indicate higher ID₅₀ values, and the data are arranged to highlight patterns of similar neutralization profiles. The data was clustered using k means, a procedure for clustering into a fixed number, k, of groups. In this procedure, the Euclidean distance between the vector of log₁₀ ID₅₀ values (i.e., the set of neutralization values in one row or column) is calculated relative to candidate group location vectors, and each serum is assigned to the cluster with the closest group location. New group means are formed on the basis of these group assignments, and this procedure is iterated until group identities do not change. This procedure was iterated 20,000 times with random initial mean vectors to find the most compact clusters. The same strategy was used to organize the isolates into serological susceptibility patterns. We added two statistical measures to assess how robust the clusters were and to determine how many clusters (k) were statistically supported in the data. To assess the impact of limited sampling, bootstrap analysis was performed by sampling the rows (or columns) with replacement 10,000 times and obtaining the fraction of times the serum (or isolate) belonged to the same cluster. The resulting degree of consensus is shown in the row or column labeled “Bootstrap” in Fig. ​Fig.2.2. To assess the impact of assay-to-assay variability, experimental “noise” was modeled from experimental data. Replicate ID₅₀ values were log₁₀ transformed, then normalized by subtracting the per-isolate or per-serum geometric means, yielding a normal distribution with a standard deviation of 0.166. Values were sampled from the distribution and added to the real data, then the k means process was repeated 1,000 times. Stability of categories for these data is shown in the row and column labeled “Noise” in Fig. ​Fig.2.2. Stability of categories for these data is shown in the row and column labeled “Noise” in Fig. ​Fig.2.2. Three clusters (k = 3) was the maximum number such that each cluster was comprised only of serum (or isolates) that were assigned to that cluster at a consistency of more than 90% by both measures of stability. Thus, each cluster shown in the boxes in Fig. ​Fig.22 represents a relatively robust grouping that would be expected to be preserved upon repeated experiments, or if different but comparable sets of sera or isolates were studied, and provides groupings of similar neutralization profiles for both sera and isolates.Open in a separate windowFIG. 2.Heat map and clustering analysis of serum. ID₅₀ values of 103 sera against 20 isolates are shown. Each row of the heat map shows ID₅₀ values for a single serum, and columns show virus isolates. Darker colors represent stronger neutralization (see key). The vertical order of sera is based on geometric mean titer; placement of clusters within this ranking uses the mean titer for all cluster members. The bars labeled “Bootstrap” or “Noise” show the results of statistical analysis of clustering. Both are visualized by mixing red, yellow, and blue corresponding to the relative frequencies of matched group assignments. A bright red, yellow, or blue color is a categorization that is unambiguous. Sera or isolates are grouped if they have a categorization of 90% or greater consistency by both the bootstrap and noise tests. Boxes highlight the clusters. Sera in red type are from LTNP. Clusters of patient sera are labeled P1, P2, and P3, while clusters of isolates are labeled I1, I2, and I3.Serum cluster P1 included the majority of LTNP sera and a few additional low potency/breadth sera (Fig. ​(Fig.2).2). Serum cluster P3 included 24 sera with the greatest potency and breadth of neutralization. In addition to showing sera and isolate k means clusters, we ordered the columns and rows in the heat map according to their geometric mean values to better visualize like patterns in the columns and rows. Five sera have higher geometric means than serum cluster 3 so are placed at the bottom of Fig. ​Fig.2.2. Among the sera with intermediate activity, one robust cluster was defined as follows: sera in this cluster (P2) do not neutralize the isolates most difficult to neutralize (geometric mean ID₅₀, 18 for P2 sera versus I3 isolates) but do neutralize isolates in the other two isolate clusters (geometric mean titers 297 and 110 for P2 sera versus I3 and I2, respectively). Thus, the patient clusters are defined not only by overall breadth or potency, but also by which isolates are neutralized. Of the clinical variables measured, only viral load was significantly associated with membership in a cluster: median VL is lower in patients with sera in cluster P1, which contains most of the LTNP, than in those with non-P1 sera (Bonferroni corrected P < 0.001).Figure ​Figure22 also shows that the panel of 20 diverse viral isolates we used to study breadth could be categorized into three clusters. Isolate cluster 3 (I3) consists of two B clade Envs, JRFL and BaL.01, which are the most neutralization-sensitive in the panel. Each of the other two clusters contains isolates of different clades. Four of five clade C isolates are in the most resistant cluster I1; these isolates are known to be sensitive to clade C sera but more resistant to clade B sera (11, 17). Of note, genetically closely related viruses were not always found within the same neutralization-susceptibility cluster. For example, isolates Q769.d22 and Q769.h5, both clade A, contain two different envelope proteins from the same patient but do not appear in the same cluster. These isolates are known to have differing sensitivities to autologous plasma and to MAb (6).We also analyzed neutralization titers of soluble CD4 (sCD4) and the commonly used monoclonal antibodies (MAb) 2G12, 4E10, 2F5, b12, and 447-52d against the same 20 isolates. The epitopes targeted by these MAb are well defined (7). It was possible that the isolate clustering of sera shown in Fig. ​Fig.22 was driven by responses that are directed mainly to epitopes thought to confer neutralization breadth. If this were the case, we hypothesized that commonalities between neutralization mediated by MAb and by sera might be observed. Clustering analysis of MAbs (Fig. ​(Fig.3)3) was performed as described above. The clustering based on patterns of neutralization by MAbs was less statistically robust than that calculated with serum titers, with no clusters found at 90% bootstrap confidence. Figure ​Figure33 shows the three clusters of isolates that appeared in only 75% of bootstrap and noise replicates. These isolate clusters (with the exception of the sensitive JRFL/Bal.01 cluster I3) were completely interspersed with those defined in the serum analysis. To further examine the relationship of serum and MAb reactivity, we compared membership of an Env in an isolate cluster as defined by serum titers (Fig. ​(Fig.2)2) with its sensitivity to each individual MAb. While membership in isolate cluster I3 correlated with neutralization titers of two of the MAb, b12 and 447-52d (P < 0.01 for each for both JRFL and BaL.01), membership in clusters I1 and I2 as defined by serum titers did not correlate with sensitivity to any one MAb. These data suggest that the clustering based on serum titers was not defined by any single epitope matching the MAb specificities.Open in a separate windowFIG. 3.Heat map and clustering analysis of monoclonal antibodies and sCD4. Each row of the heat map shows IC₅₀ values for a single reagent, and columns show virus isolates. See legend to Fig. ​Fig.22 for an explanation. Boxes show isolates assigned to clusters at the 75% level. Neutralization data are from references 16 and 17 and this study.This clustering analysis allowed us to discern patterns of neutralization reactivity that are distinct from clade and from sensitivity to known cross-neutralizing MAbs. The appearance of isolates from multiple clades in clusters I1 and I2 is consistent with prior analyses of MAbs (5) and sera (11, 14) in which sequences from the same clade were distributed among multiple clusters. In general, the clade of a virus does not predict its sensitivity to patient sera and is not directly equivalent to a neutralization serotype (13, 21, 26, 34). Furthermore, the discordance between the results for MAbs and for sera may suggest that the clustering based on serum titers was not dominated by reactivities that are similar to those of the MAbs. It is unclear at present whether these differences are mediated by targeting of conserved epitopes that are not yet identified, epitopes similar to those of MAbs but with differences in neutralization patterns, or multiple specificities. These findings are potentially consistent with antibody cloning (30) and serum mapping (4, 8, 11, 18, 19, 29) studies which found that in some sera, multiple specificities are responsible for the breadth of neutralization. Future studies of this cohort using additional isolates may allow determination of possible neutralization serotypes, and viral sequence motifs that are signatures of neutralization cluster, as for the clade C sera analyzed in reference 14, or neutralization sensitivity.The clinical data suggest that extended exposure to antigen may be beneficial for the development of broad NAbs. We observed that viral load has a modest positive correlation with neutralization breadth (Fig. ​(Fig.1C),1C), as was also seen in cohorts in the United States (29) and Kenya (28). Conversely, LTNP with a VL of <50 rarely had breadth (Fig. ​(Fig.1B),1B), again consistent with other reports (1, 27). Length of exposure also seems to play a role in the development of broad NAbs: Sather et al. (29) noted an association of breadth with the duration of infection in a seroconversion cohort. These associations are modest, and several slow progressors had low NAbs; thus, antigen may be necessary but not sufficient for the development of broad NAbs. Long-term exposure to HIV antigen has been shown to directly impact immunoglobulin G (IgG) development: Scheid et al. (30) found that Env-specific IgG genes were highly mutated compared to other IgG genes in patients with broad NAbs, implying multiple rounds of selection and hypermutation in response to persistence or turnover of viral antigen. Collectively, these data suggest that a vaccine may need to supply viral antigen for long periods of time, via multiple dosing or a replication-competent vector, to allow antibody maturation and development of a broad neutralizing response.The prevalence and titers of NAbs in chronically HIV-infected patients provide encouragement for the development of vaccines that elicit protective humoral immunity. We found that that 20% of progressor patients make broad NAbs; although our cohort is enriched for slow progressors, similar frequencies were noted in other, less-selected cohorts (11, 29, 32). The fact that so many patients make broad NAbs, even in the setting of B-cell dysfunction caused by HIV (25), demonstrates the ability of the human immune system to generate such NAbs. An appropriate vaccine given to immunocompetent individuals could potentially elicit broad NAbs at higher frequencies. Furthermore, most patients neutralized at least some isolates with titers in the hundreds in the TZM-bl assay. Data from a recent passive transfer experiment in a low-dose-challenge simian-HIV (SHIV) macaque model demonstrated a protective effect of neutralizing titers of 1:200 in the TZM-bl assay (12). Thus, these examples show that it is possible to elicit NAbs at sufficient levels and breadth to potentially contribute to the protective efficacy of an HIV vaccine.     

Role of Glycosaminoglycan Sulfation in the Formation of Immunoglobulin Light Chain Amyloid Oligomers and Fibrils

Primary amyloidosis (AL) results from overproduction of unstable monoclonal immunoglobulin light chains (LCs) and the deposition of insoluble fibrils in tissues, leading to fatal organ disease. Glycosaminoglycans (GAGs) are associated with AL fibrils and have been successfully targeted in the treatment of other forms of amyloidosis. We investigated the role of GAGs in LC fibrillogenesis. Ex vivo tissue amyloid fibrils were extracted and examined for structure and associated GAGs. The GAGs were detected along the length of the fibril strand, and the periodicity of heparan sulfate (HS) along the LC fibrils generated in vitro was similar to that of the ex vivo fibrils. To examine the role of sulfated GAGs on AL oligomer and fibril formation in vitro, a κ1 LC purified from urine of a patient with AL amyloidosis was incubated in the presence or absence of GAGs. The fibrils generated in vitro at physiologic concentration, temperature, and pH shared morphologic characteristics with the ex vivo κ1 amyloid fibrils. The presence of HS and over-O-sulfated-heparin enhanced the formation of oligomers and fibrils with HS promoting the most rapid transition. In contrast, GAGs did not enhance fibril formation of a non-amyloidogenic κ1 LC purified from urine of a patient with multiple myeloma. The data indicate that the characteristics of the full-length κ1 amyloidogenic LC, containing post-translational modifications, possess key elements that influence interactions of the LC with HS. These findings highlight the importance of the variable and constant LC regions in GAG interaction and suggest potential therapeutic targets for treatment.     

Differential CMV-specific CD8+ effector T cell responses in the lung allograft predominate over the blood during human primary infection

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient- (D+R-) individuals. In 15 D+R- LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA- during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA- phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.     

c-Jun N-terminal kinase specifically phosphorylates p66ShcA at serine 36 in response to ultraviolet irradiation

Mice lacking expression of the p66 isoform of the ShcA adaptor protein (p66(ShcA)) are less susceptible to oxidative stress and have an extended life span. Specifically, phosphorylation of p66(ShcA) at serine 36 is critical for the cell death response elicited by oxidative damage. We sought to identify the kinase(s) responsible for this phosphorylation. Utilizing the SH-SY5Y human neuroblastoma cell model, it is demonstrated that p66(ShcA) is phosphorylated on serine/threonine residues in response to UV irradiation. Both c-Jun N-terminal kinases (JNKs) and p38 mitogen-activated protein kinases are activated by UV irradiation, and we show that both are capable of phosphorylating serine 36 of p66(ShcA) in vitro. However, treatment of cells with a multiple lineage kinase inhibitor, CEP-1347, that blocks UV-induced JNK activation, but not p38, phosphatidylinositol 3-kinase, or MEK1 inhibitors, prevented p66(ShcA) phosphorylation in SH-SY5Y cells. Consistent with this finding, transfected activated JNK1, but not the kinase-dead JNK1, leads to phosphorylation of serine 36 of p66(ShcA) in Chinese hamster ovary cells. In conclusion, JNKs are the kinases that phosphorylate serine 36 of p66(ShcA) in response to UV irradiation in SH-SY5Y cells, and blocking p66(ShcA) phosphorylation by intervening in the JNK pathway may prevent cellular damage due to light-induced oxidative stress.     

Evidence of IL-18 as a novel angiogenic mediator

Angiogenesis, or new blood vessel growth, is a key process in the development of synovial inflammation in rheumatoid arthritis (RA). Integral to this pathologic proliferation are proinflammatory cytokines. We hypothesized a role for IL-18 as an angiogenic mediator in RA. We examined the effect of human IL-18 on human microvascular endothelial cell (HMVEC) migration. IL-18 induced HMVEC migration at 1 nM (p < 0.05). RA synovial fluids potently induced endothelial cell migration, but IL-18 immunodepletion resulted in a 68 +/- 5% decrease in HMVEC migration (p < 0.05). IL-18 appears to act on HMVECs via alpha(v)beta(3) integrin. To test whether IL-18 induced endothelial cell tube formation in vitro, we quantitated the degree of tube formation on Matrigel matrix. IL-18, 1 or 10 nM, resulted in a 77% or 87% increase in tube formation compared with control (p < 0.05). To determine whether IL-18 may be angiogenic in vivo, we implanted IL-18 in Matrigel plugs in mice, and IL-18 at 1 and 10 nM induced angiogenesis (p < 0.05). The angiogenesis observed appears to be independent of the contribution of local TNF-alpha, as evidenced by adding neutralizing anti-TNF-alpha Ab to the Matrigel plugs. In an alternative in vivo model, sponges embedded with IL-18 or control were implanted into mice. IL-18 (10 nM) induced a 4-fold increase in angiogenesis vs the control (p < 0.05). These findings support a novel function for IL-18 as an angiogenic factor in RA and may elucidate a potential therapeutic target for angiogenesis-directed diseases.     

Betabellins 15D and 16D, de Novo designed beta-sandwich proteins that have amyloidogenic properties

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta-sheets packed against one another by hydrophobic interactions. d-Amino acid residues are used to energetically favor formation of type-I' beta turns. Air oxidation of betabellin 15S (B15S) (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p denotes d-Pro, h denotes d-His, and k denotes d-Lys) yields betabellin 15D (B15D), a 64-residue disulfide-bridged protein. The amino acid sequence of B15D contains a conformationally constrained d-Pro residue at the i + 1 position of each type-I' beta turn. To test whether d-Pro residues are necessary for folding at these positions, the six d-Pro residues of B15D are replaced by d-Ala residues in betabellin 16D (B16D). Previously, transmission electron microscopy showed that B15D forms unbranched, 35-A wide fibrils that associate into bundles in 5.0 mM 3-(N-morpholino)propanesulfonate and 250 mM NaCl at pH 7; under these conditions, B16D forms ribbon-like assemblies. The B15D fibrils resemble the protofilaments that constitute amyloid fibrils. The present studies show that both B15D and B16D have characteristics of amyloidogenic proteins: the unbranched fibrils and ribbons stained with Congo red and displayed a green birefringence, exhibited a cross-beta structure, and bound 1-anilino-8-naphthalenesulfonate. Thus, these de novo designed beta-sandwich proteins should provide useful models for studying the mechanism of amyloid protofilament formation and assembly into amyloid fibrils and for designing potential inhibitors of amyloidogenesis.